|
| Status |
Public on Mar 25, 2009 |
| Title |
H3K27me3 ChIP in Ezh2-/- cells on G chip |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K27me3 ChIP in Ezh2-/- cells (3 replicates)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem
antibody: H3K27me3
cell line: CJ7
genotype: Ezh2-/-
|
| Treatment protocol |
wild-type (WT) and Ezh2-/- mutant ES cells were fixed with 0.8% formaldehyde for 10 min at room temperature. Cell lydates were ChIP'ed by antibody recognizing H3K27me3 mark (Upstate) or by using streptavidin-Dyna beads (Invitrogen). DNA fragments were isolated and amplified by LM-PCR. Amplified DNA was fragmented and biotin labeled before hybridiztion.
|
| Growth protocol |
standard culture conditions were used to culture ES cells.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
pulled down chromatin was reversely crosslinked and DNA was precipitated by isopropanol.
|
| Label |
biotin
|
| Label protocol |
Fragmented DNA was labeled with biotin-N6-ddATP by terminal deoxynucleiotidyl transferase.
|
| |
|
| Channel 2 |
| Source name |
INPUT CONTROL
|
| Organism |
Mus musculus |
| Characteristics |
input 1: genomic DNA from J1 cells expressing bfEZH1
input 2: genomic DNA from CJ7 cells
input 3: genomic DNA from Ezh2-/- cells
|
| Treatment protocol |
wild-type (WT) and Ezh2-/- mutant ES cells were fixed with 0.8% formaldehyde for 10 min at room temperature. Cell lydates were ChIP'ed by antibody recognizing H3K27me3 mark (Upstate) or by using streptavidin-Dyna beads (Invitrogen). DNA fragments were isolated and amplified by LM-PCR. Amplified DNA was fragmented and biotin labeled before hybridiztion.
|
| Growth protocol |
standard culture conditions were used to culture ES cells.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
pulled down chromatin was reversely crosslinked and DNA was precipitated by isopropanol.
|
| Label |
biotin
|
| Label protocol |
Fragmented DNA was labeled with biotin-N6-ddATP by terminal deoxynucleiotidyl transferase.
|
| |
|
| |
| Hybridization protocol |
About 2-4 ug of labeled DNA were hybridized for 16 hr at 45C on tiling arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
ChIP-chip
|
| Data processing |
The data were analyzed with Model-based Analysis of Tiling-arrays for ChIP-chip (MAT) developed by the Liu lab and processed based on MM8 annotation. MATScore, p value (in form of -10log10_Pvalue), fold-change, FDR, peak position and length were reported for each peak identified by MAT program (results provided as supplementary text files).
|
| |
|
| Submission date |
Mar 25, 2009 |
| Last update date |
Mar 25, 2009 |
| Contact name |
Xiaohua Shen |
| E-mail(s) |
shen@bloodgroup.tch.harvard.edu
|
| Phone |
617-632-6496
|
| Organization name |
Dana Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Lab |
Stuart Orkin lab
|
| Street address |
44 Binney Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL6450 |
| Series (1) |
| GSE15388 |
EZH1 and H3K27me3 ChIP-chip data from mouse embryonic stem cells |
|
